A-Kinase Anchoring Proteins constitute a diverse family of scaffold proteins which share a common binding site for the RI/II subunit of PKA and are now recognized as scaffolds for multivalent cell signaling. AKAP250 (a.k.a., AKAP12, gravin, or a human homologue of SSeCKS) plays, a critical role in signaling of the beta2-adrenergic receptor (beta2AR), the prototype for the superfamily of G-protein-coupled receptors (GPCR). We have demonstrated that AKAP250 is multivalent, providing a scaffold for beta2AR signaling complexes that include minimally PKA, PKC, PP2B, Src, and the beta2AR. Agonist activation of the beta2AR stimulates event desensitization, sequestration, followed by resensitization, and recycling of the receptor, a process disrupted in the absence of AKAP250. Recently we have mapped the domains of the C-terminal tail of the beta2AR as well as those AKAP motifs of AKAP250 that provide the basis for protein-protein interactions of the scaffold-receptor and revealed a central role of protein phosphorylation of both partners in defining this dynamic interaction. The overarching goal of this research plan is to understand at the "meso"-scale, the dynamic role of AKAP scaffold in beta2AR signaling complexes. Four specific aims target the overarching goal: namely 1) to probe protein-lipid and protein-protein interactions of AKAP250 in beta2AK signaling (focusing on three domains that may dictate scaffold-membrane association, i.e. N-myristoylation, MARCKS protein-like Membrane Effector Domain, and beta2AR-interacting domain of the scaffold, 2) to identify new signaling molecules that constitute AKAP-based beta2AR signaling complexes and to establish their function in the complex (making use of yeast-2-hybrid, SOS recruitment system yeast 2-hybrid, and HTS proteomic analyses of complex pull-downs using LC/MS/MS); 3) to .map the spatial organization and trafficking mechanisms beta2AR signaling complexes in response to stimulation by beta-agonist (desensitization) and by insulin (counterregulatlon), using multi-photon confocal microscopy in tandem with organelle-specific markers as well as bioluminescence resonance energy transfer (BRET) spectroscopy; and 4) to elucidate the ordered pattern of phosphorylation of key molecules and their individual residues constituting AKAP-based beta2 AR signaling complexes and its function in the dynamic regulation of the complexes (using LC/MS/MS in tandem with domain-specific mutagenesis). The targeted convergence of mass spectrometry-based proteomics, sensitive multi-photon confocal microscopy, BRET spectroscopy, and read-outs of cellular (organelle-specific) localization, function, and protein-protein interactions create an unparalleled opportunity for success. Establishing the key roles of AKAP scaffolds in GPCR signaling is essential for our understanding of GPCR in normal and disease states as well as for developing novel therapeutic interventions.